UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

Kol Jia Yong

A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems
Kazlauskiene M, Kostiuk G, Venclovas Č, Tamulaitis G, Siksnys V.
Science. 2017 Aug 11;357(6351):605-609. doi: 10.1126/science.aao0100. Epub 2017 Jun 29.
August 11, 2017
Institute of Biotechnology, Vilnius University, Saulėtekio Avenue 7, 10257 Vilnius, Lithuania. Institute of Biotechnology, Vilnius University, Saulėtekio Avenue 7, 10257 Vilnius, Lithuania. [email protected] [email protected]
Type III CRISPR-Cas systems in prokaryotes provide immunity against invading nucleic acids through the coordinated degradation of transcriptionally active DNA and its transcripts by the Csm effector complex. The Cas10 subunit of the complex contains an HD nuclease domain that is responsible for DNA degradation and two Palm domains with elusive functions. In addition, Csm6, a ribonuclease that is not part of the complex, is also required to provide full immunity. We show here that target RNA binding by the Csm effector complex of Streptococcus thermophilus triggers Cas10 to synthesize cyclic oligoadenylates (cA n ; n = 2 to 6) by means of the Palm domains. Acting as signaling molecules, cyclic oligoadenylates bind Csm6 to activate its nonspecific RNA degradation. This cyclic oligoadenylate-based signaling pathway coordinates different components of CRISPR-Cas to prevent phage infection and propagation.
Date: 
September 20, 2017
Where: 
HSW 1057 at noon

Gabriel Eades

A heterochromatin-dependent transcription machinery drives piRNA expression
Peter Refsing Andersen, Laszlo Tirian, Milica Vunjak & Julius Brennecke.
Nature. 2017 Sep 7;549(7670):54-59. doi: 10.1038/nature23482. Epub 2017 Aug 23.
August 23, 2017
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Dr. Bohrgasse 3, 1030 Vienna, Austria.
Nuclear small RNA pathways safeguard genome integrity by establishing transcription-repressing heterochromatin at transposable elements. This inevitably also targets the transposon-rich source loci of the small RNAs themselves. How small RNA source loci are efficiently transcribed while transposon promoters are potently silenced is not understood. Here we show that, in Drosophila, transcription of PIWI-interacting RNA (piRNA) clusters—small RNA source loci in animal gonads—is enforced through RNA polymerase II pre-initiation complex formation within repressive heterochromatin. This is accomplished through Moonshiner, a paralogue of a basal transcription factor IIA (TFIIA) subunit, which is recruited to piRNA clusters via the heterochromatin protein-1 variant Rhino. Moonshiner triggers transcription initiation within piRNA clusters by recruiting the TATA-box binding protein (TBP)-related factor TRF2, an animal TFIID core variant. Thus, transcription of heterochromatic small RNA source loci relies on direct recruitment of the core transcriptional machinery to DNA via histone marks rather than sequence motifs, a concept that we argue is a recurring theme in evolution.
Date: 
September 13, 2017
Where: 
HSW 1057 at noon

Theodore Roth

In Situ Capture of Chromatin Interactions by Biotinylated dCas9
Liu X1, Zhang Y1, Chen Y2, Li M3, Zhou F4, Li K1, Cao H1, Ni M1, Liu Y1, Gu Z1, Dickerson KE1, Xie S5, Hon GC5, Xuan Z2, Zhang MQ6, Shao Z3, Xu J7.
Cell. 2017 Aug 24;170(5):1028-1043.e19. doi: 10.1016/j.cell.2017.08.003.
August 24, 2017
Children's Medical Center Research Institute, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Department of Biological Sciences, Center for Systems Biology, University of Texas at Dallas, Richardson, TX 75080, USA. Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. Liver Cancer Institute, Zhongshan Hospital, Key Laboratory of Carcinogenesis and Cancer Invasion, Minister of Education, and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China. Electronic address: [email protected] Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Department of Biological Sciences, Center for Systems Biology, University of Texas at Dallas, Richardson, TX 75080, USA; MOE Key Laboratory of Bioinformatics; Bioinformatics Division and Center for Synthetic and Systems Biology, TNLIST; Department of Automation, Tsinghua University, Beijing 100084, China. Children's Medical Center Research Institute, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address: [email protected]
Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here, we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single-copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes establishes evidence for composition-based hierarchical organization. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally regulated super-enhancers reveals spatial features that causally control gene transcription. Thus, comprehensive and unbiased analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.
Date: 
September 6, 2017
Where: 
HSW 1057 at noon

Eleonora De Klerk

Chromatin-enriched lncRNAs can act as cell-type specific activators of proximal gene transcription
Werner MS1, Sullivan MA1,2, Shah RN1,2, Nadadur RD3, Grzybowski AT1, Galat V4, Moskowitz IP3, Ruthenburg AJ1,2.
Nat Struct Mol Biol. 2017 Jul;24(7):596-603. doi: 10.1038/nsmb.3424. Epub 2017 Jun 19.
July 1, 2017
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois, USA. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois, USA. Department of Pediatrics and Pathology, The University of Chicago, Chicago, Illinois, USA. Department of Pathology, Stanley Manne Children's Research Institute, Ann and Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
We recently described a new class of long noncoding RNAs (lncRNAs) that are distinguished by especially tight chromatin association and whose presence is strongly correlated to expression of nearby genes. Here, we examine the cis-enhancer mechanism of this class of chromatin-enriched RNA (cheRNA) across multiple human cell lines. cheRNAs are largely cell type specific and provide the most reliable chromatin signature to predict cis-gene transcription in every human cell type examined. Targeted depletion of three cheRNAs decreases expression of their neighboring genes, indicating potential co-activator function, and single-molecule fluorescence in situ hybridization (smFISH) of one cheRNA-distal target gene pair suggests a spatial overlap consistent with a role in chromosome looping. Additionally, the cheRNA HIDALGO stimulates the fetal hemoglobin subunit gamma 1 (HBG1) gene during erythroid differentiation by promoting contacts to a downstream enhancer. Our results suggest that multiple cheRNAs activate proximal lineage-specific gene transcription.
Date: 
August 30, 2017
Where: 
HSW 1057 at noon

Malin Akerblom

Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function
Piwecka M1, Glažar P1, Hernandez-Miranda LR2, Memczak S1,3, Wolf SA4, Rybak-Wolf A1, Filipchyk A1, Klironomos F1, Cerda Jara CA1, Fenske P5, Trimbuch T5, Zywitza V1, Plass M1, Schreyer L1, Ayoub S1, Kocks C1, Kühn R6,7, Rosenmund C5, Birchmeier C2, Rajewsky N8.
Science. 2017 Aug 10. pii: eaam8526. doi: 10.1126/science.aam8526. [Epub ahead of print]
August 10, 2017
Laboratory for Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin-Buch, Germany. Laboratory for Developmental Biology and Signal Transduction, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin-Buch, Germany. Experimental and Clinical Research Center, a joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine, Berlin, Germany. Laboratory for Cellular Neurosciences, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin-Buch, Germany. Department of Neurophysiology, NeuroCure Cluster of Excellence, Charité-Universitätsmedizin, Berlin, Germany. Transgenic Core Facility, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin-Buch, Germany. Berlin Institute of Health, Kapelle-Ufer 2, Berlin, Germany. Laboratory for Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin-Buch, Germany. [email protected]
Hundreds of circular RNAs (circRNAs) are highly abundant in mammalian brain, with oftentimes conserved expression. Here, we show that the circRNA Cdr1as is massively bound by miR-7 and miR-671 in the human and mouse brain. When the Cdr1as locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating, a deficit in the ability to filter out unnecessary information associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of microRNAs miR-7 and miR-671 was specifically and post-transcriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as Fos, a direct miR-7 target, was enhanced in Cdr1as-deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between circRNAs and miRNAs are important for normal brain function.
Date: 
August 23, 2017
Where: 
HSW 1057 at noon

Bin Zhang

Structural basis of CRISPR–SpyCas9 inhibition by an anti-CRISPR protein
Dong1, Guo M1, Wang S1, Zhu Y1, Wang S1, Xiong Z1, Yang J1, Xu Z1, Huang Z1.
Nature. 2017 Jun 15;546(7658):436-439. doi: 10.1038/nature22377. Epub 2017 Apr 27.
June 15, 2017
HIT Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
CRISPR-Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a protospacer adjacent motif (PAM) and sequences complementary to the guide RNA. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing 'off-switch' tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.
Date: 
August 16, 2017
Where: 
HSW 1057 at noon

Vanille Greiner

Correction of a pathogenic gene mutation in human embryos
Hong Ma1*, Nuria Marti-Gutierrez1*, Sang-Wook Park2*, Jun Wu3*, Yeonmi Lee1, Keiichiro Suzuki3, Amy Koski1, Dongmei Ji1, Tomonari Hayama1, Riffat Ahmed1, Hayley Darby1, Crystal Van Dyken1, Ying Li1, Eunju Kang1, A.-Reum Park2, Daesik Kim4, Sang-Tae Kim2, Jianhui Gong5,6,7,8, Ying Gu5,6,7, Xun Xu5,6,7, David Battaglia1,9, Sacha A. Krieg9, David M. Lee9, Diana H. Wu9, Don P. Wolf1, Stephen B. Heitner10, Juan Carlos Izpisua Belmonte3§, Paula Amato1,9§, Jin-Soo Kim2,4§, Sanjiv Kaul10§ & Shoukhrat Mitalipov1,10§
Nature. Received: March 28, 2017. Accepted June 27, 2017. Published online August 2, 2017.
August 2, 2017
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA. 2Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon, 34047, Republic of Korea. 3Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA. 4Department of Chemistry, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul, 151-747, Republic of Korea. 5BGI-Shenzhen, Build 11, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China. 6China National GeneBank, BGI-Shenzhen, Jinsha Road, Dapeng District, Shenzhen, 518210, China. 7BGI-Qingdao, 2877 Tuanjie Road, Sino- German Ecopark, Qingdao, 266000, China. 8Shenzhen Engineering Laboratory for Innovative Molecular Diagnostics, BGI-Shenzhen, Build 11, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China. 9Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA. 10Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road, Portland, Oregon 97239, USA. * These authors contributed equally to this work. §These authors jointly supervised this work.
Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR–Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.
Date: 
August 9, 2017
Where: 
HSW 1057 at noon

Roman Camarda

Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer
Barzin Y. Nabet, Yu Qiu, Jacob E. Shabason, Tony J. Wu, Taewon Yoon, Brian C. Kim, Joseph L. Benci, Angela M. DeMichele, Julia Tchou, Joseph Marcotrigiano, and Andy J. Minn
Cell
July 27, 2017
Department of Radiation Oncology Department of Medicine Department of Surgery Institute for Immunology Parker Institute for Cancer Immunotherapy Basser Center for BRCA Abramson Cancer Center Abramson Family Cancer Research Institute Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA
Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer.
Date: 
August 2, 2017
Where: 
HSW 1057 at noon

Ryan Wagner

TBA
Date: 
July 26, 2017
Where: 
HSW 1057 at noon

Gabriel Eades

TERRA RNA Antagonizes ATRX and Protects Telomeres
Hsueh-Ping Chu, Catherine Cifuentes-Rojas, Barry Kesner, Eric Aeby, Hun-goo Lee, Chunyao Wei, Hyun Jung Oh, Myriam Boukhali, Wilhelm Haas, Jeannie T. Lee
Cell
June 29, 2017
1Howard Hughes Medical Institute, Boston, MA 02114, USA 2Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA 3Department of Genetics, Harvard Medical School, Boston, MA 02114, USA 4Massachusetts General Hospital Cancer Center, Charlestown, Boston, MA 02114, USA 5Department of Medicine, Harvard Medical School, Boston, MA 02114, USA
Through an integration of genomic and proteomic approaches to advance understanding of long noncoding RNAs, we investigate the function of the telomeric transcript, TERRA. By identifying thousands of TERRA target sites in the mouse genome, we demonstrate that TERRA can bind both in cis to telomeres and in trans to genic targets. We then define a large network of interacting proteins, including epigenetic factors, telomeric proteins, and the RNA helicase, ATRX. TERRA and ATRX share hundreds of target genes and are functionally antagonistic at these loci: whereas TERRA activates, ATRX represses gene expression. At telomeres, TERRA competes with telomeric DNA for ATRX binding, suppresses ATRX localization, and ensures telomeric stability. Depleting TERRA increases telomerase activity and induces telomeric pathologies, including formation of telomere-induced DNA damage foci and loss or duplication of telomeric sequences. We conclude that TERRA functions as an epigenomic modulator in trans and as an essential regulator of telomeres in cis.
Date: 
July 19, 2017
Where: 
HSW 1057 at noon